Molecular Characterization and Prospective Evaluation of Pathologic Response and Outcomes with Neoadjuvant Therapy in Metaplastic Triple-Negative Breast Cancer.

PURPOSE: Metaplastic breast cancer (MpBC) is a rare subtype of breast cancer that is commonly triple-negative and poorly responsive to neoadjuvant therapy in retrospective studies. EXPERIMENTAL DESIGN: To better define clinical outcomes and correlates of response, we analyzed the rate of pathologic complete response (pCR) to neoadjuvant therapy, survival outcomes, and genomic and transcriptomic profiles of the pretreatment tumors in a prospective clinical trial (NCT02276443). A total of 211 patients with triple-negative breast cancer (TNBC), including 39 with MpBC, received doxorubicin-cyclophosphamide-based neoadjuvant therapy. RESULTS: Although not meeting the threshold for statistical significance, patients with MpBCs were less likely to experience a pCR (23% vs. 40%; P = 0.07), had shorter event-free survival (29.4 vs. 32.2 months, P = 0.15), metastasis-free survival (30.3 vs. 32.4 months, P = 0.22); and overall survival (32.6 vs. 34.3 months, P = 0.21). This heterogeneity is mirrored in the molecular profiling. Mutations in PI3KCA (23% vs. 9%, P = 0.07) and its pathway (41% vs. 18%, P = 0.02) were frequently observed and enriched in MpBCs. The gene expression profiles of each histologically defined subtype were distinguishable and characterized by distinctive gene signatures. Among nonmetaplastic (non-Mp) TNBCs, 10% possessed a metaplastic-like gene expression signature and had pCR rates and survival outcomes similar to MpBC. CONCLUSIONS: Further investigations will determine if metaplastic-like tumors should be treated more similarly to MpBC in the clinic. The 23% pCR rate in this study suggests that patients with MpBC should be considered for NAT. To improve this rate, a pathway analysis predicted enrichment of histone deacetylase (HDAC) and RTK/MAPK pathways in MpBC, which may serve as new targetable vulnerabilities.

Male Mammary Paget Disease: A Tale of 2 Contrasting Cases.

Mammary Paget disease (MPD) comprises 1.45% all male breast cancers, compared with only 0.68% of all female breast cancers. Patients usually present in the fifth and sixth decades of life with ulceration, eczematous changes, discharge, bleeding, itching, and induration of the nipple and areola. Typically, there is a delay in definitive diagnosis and treatment from the onset of symptoms because most patients are initially treated for a rash. At the time of diagnosis, about half of the patients may have palpable breast mass, positive lymph nodes, or both. In this article, we present 2 cases of male MPD representing the extremes of clinical, radiologic, and histopathologic spectrum of the disease. One patient presented with a rash of the nipple of several months duration without an underlying lesion, whereas the other presented with sensitivity and pain of the nipple for 1 year and an underlying mass. Biopsies were diagnostic of MPD in both cases, and definitive surgery revealed an underlying ductal carcinoma in situ in the first case and an invasive ductal carcinoma in the second, highlighting the importance of early biopsy to initiate appropriate management.

Cell-free Circulating Tumor DNA Variant Allele Frequency Associates with Survival in Metastatic Cancer.

PURPOSE: Physicians are expected to assess prognosis both for patient counseling and for determining suitability for clinical trials. Increasingly, cell-free circulating tumor DNA (cfDNA) sequencing is being performed for clinical decision making. We sought to determine whether variant allele frequency (VAF) in cfDNA is associated with prognosis. EXPERIMENTAL DESIGN: We performed a retrospective analysis of 298 patients with metastatic disease who underwent clinical comprehensive cfDNA analysis and assessed association between VAF and overall survival. RESULTS: cfDNA mutations were detected in 240 patients (80.5%). Median overall survival (OS) was 11.5 months. cfDNA mutation detection and number of nonsynonymous mutations (NSM) significantly differed between tumor types, being lowest in appendiceal cancer and highest in colon cancer. Having more than one NSM detected was associated with significantly worse OS (HR = 2.3; P < 0.0001). VAF was classified by quartiles, Q1 lowest, Q4 highest VAF. Higher VAF levels were associated with a significantly worse overall survival (VAF Q3 HR 2.3, P = 0.0069; VAF Q4 HR = 3.8, P < 0.0001) on univariate analysis. On multivariate analysis, VAF Q4, male sex, albumin level <3.5 g/dL, number of nonvisceral metastatic sites >0 and number of prior therapies >4 were independent predictors of worse OS. CONCLUSIONS: Higher levels of cfDNA VAF and a higher number of NSMs were associated with worse OS in patients with metastatic disease. Further study is needed to determine optimal VAF thresholds for clinical decision making and the utility of cfDNA VAF as a prognostic marker in different tumor types.

Akt Activation Mediates Acquired Resistance to Fibroblast Growth Factor Receptor Inhibitor BGJ398.

Activation of FGFR signaling through mutations, amplifications, or fusions involving FGFR1, 2, 3, or 4 is seen in multiple tumors, including lung, bladder, and cholangiocarcinoma. Currently, several clinical trials are evaluating the role of novel FGFR inhibitors in solid tumors. As we move forward with FGFR inhibitors clinically, we anticipate the emergence of resistance with treatment. Consequently, we sought to study the mechanism(s) of acquired resistance to FGFR inhibitors using annotated cancer cell lines. We identified cancer cell lines that have activating mutations in FGFR1, 2, or 3 and treated them chronically with the selective FGFR inhibitor, BGJ398. We observed resistance to chronic BGJ398 exposure in DMS114 (small-cell lung cancer, FGFR1 amplification) and RT112 (urothelial carcinoma, FGFR3 fusion/amplification) cell lines based on viability assays. Reverse-phase protein array (RPPA) analysis showed increased phosphorylation of Akt (T308 and S473) and its downstream target GSK3 (S9 and S21) in both the resistant cell lines when compared with matching controls. Results of RPPA were confirmed using immunoblots. Consequently, the addition of an Akt inhibitor (GSK2141795) or siRNA was able to restore sensitivity to BGJ398 in resistant cell lines. These data suggest a role for Akt pathway in mediating acquired resistance to FGFR inhibition. Mol Cancer Ther; 16(4); 614-24. ©2017 AACR.

Cancer Driver Log (CanDL): Catalog of Potentially Actionable Cancer Mutations.

Massively parallel sequencing technologies have enabled characterization of genomic alterations across multiple tumor types. Efforts have focused on identifying driver mutations because they represent potential targets for therapy. However, because of the presence of driver and passenger mutations, it is often challenging to assign the clinical relevance of specific mutations observed in patients. Currently, there are multiple databases and tools that provide in silico assessment for potential drivers; however, there is no comprehensive resource for mutations with functional characterization. Therefore, we created an expert-curated database of potentially actionable driver mutations for molecular pathologists to facilitate annotation of cancer genomic testing. We reviewed scientific literature to identify variants that have been functionally characterized in vitro or in vivo as driver mutations. We obtained the chromosome location and all possible nucleotide positions for each amino acid change and uploaded them to the Cancer Driver Log (CanDL) database with associated literature reference indicating functional driver evidence. In addition to a simple interface, the database allows users to download all or selected genes as a comma-separated values file for incorporation into their own analysis pipeline. Furthermore, the database includes a mechanism for third-party contributions to support updates for novel driver mutations. Overall, this freely available database will facilitate rapid annotation of cancer genomic testing in molecular pathology laboratories for mutations.

Evaluation of Hybridization Capture Versus Amplicon-Based Methods for Whole-Exome Sequencing.

Next-generation sequencing has aided characterization of genomic variation. While whole-genome sequencing may capture all possible mutations, whole-exome sequencing remains cost-effective and captures most phenotype-altering mutations. Initial strategies for exome enrichment utilized a hybridization-based capture approach. Recently, amplicon-based methods were designed to simplify preparation and utilize smaller DNA inputs. We evaluated two hybridization capture-based and two amplicon-based whole-exome sequencing approaches, utilizing both Illumina and Ion Torrent sequencers, comparing on-target alignment, uniformity, and variant calling. While the amplicon methods had higher on-target rates, the hybridization capture-based approaches demonstrated better uniformity. All methods identified many of the same single-nucleotide variants, but each amplicon-based method missed variants detected by the other three methods and reported additional variants discordant with all three other technologies. Many of these potential false positives or negatives appear to result from limited coverage, low variant frequency, vicinity to read starts/ends, or the need for platform-specific variant calling algorithms. All methods demonstrated effective copy-number variant calling when evaluated against a single-nucleotide polymorphism array. This study illustrates some differences between whole-exome sequencing approaches, highlights the need for selecting appropriate variant calling based on capture method, and will aid laboratories in selecting their preferred approach.

Clinical tumor sequencing: opportunities and challenges for precision cancer medicine.

Advances in tumor genome sequencing have enabled discovery of actionable alterations leading to novel therapies. Currently, there are approved targeted therapies across various tumors that can be matched to genomic alterations, such as point mutations, gene amplification, and translocations. Tools to detect these genomic alterations have emerged as a result of decreasing costs and improved throughput enabled by next-generation sequencing (NGS) technologies. NGS has been successfully utilized for developing biomarkers to assess susceptibility, diagnosis, prognosis, and treatment of cancers. However, clinical application presents some potential challenges in terms of tumor specimen acquisition, analysis, privacy, interpretation, and drug development in rare cancer subsets. Although whole-genome sequencing offers the most complete strategy for tumor analysis, its present utility in clinical care is limited. Consequently, targeted gene capture panels are more commonly employed by academic institutions and commercial vendors for clinical grade cancer genomic testing to assess molecular eligibility for matching therapies, whereas whole-exome and transcriptome (RNASeq) sequencing are being utilized for discovery research. This review discusses the strategies, clinical challenges, and opportunities associated with the application of cancer genomic testing for precision cancer medicine.

Comparison of custom capture for targeted next-generation DNA sequencing.

Targeted, capture-based DNA sequencing is a cost-effective method to focus sequencing on a coding region or other customized region of the genome. There are multiple targeted sequencing methods available, but none has been systematically investigated and compared. We evaluated four commercially available custom-targeted DNA technologies for next-generation sequencing with respect to on-target sequencing, uniformity, and ability to detect single-nucleotide variations (SNVs) and copy number variations. The technologies that used sonication for DNA fragmentation displayed impressive uniformity of capture, whereas the others had shorter preparation times, but sacrificed uniformity. One of those technologies, which uses transposase for DNA fragmentation, has a drawback requiring sample pooling, and the last one, which uses restriction enzymes, has a limitation depending on restriction enzyme digest sites. Although all technologies displayed some level of concordance for calling SNVs, the technologies that require restriction enzymes or transposase missed several SNVs largely because of the lack of coverage. All technologies performed well for copy number variation calling when compared to single-nucleotide polymorphism arrays. These results enable laboratories to compare these methods to make informed decisions for their intended applications.

From single-gene to multiplex analysis in lung cancer, challenges and accomplishments: a review of a single institution's experience.

Molecular selection has led to the successful use of novel tyrosine kinase inhibitors (TKIs) in non-small-cell lung cancers (NSCLCs). For instance, mutations in EGFR and translocations and fusions in ALK render tumor cells sensitive to some TKIs, leading to substantial clinical benefits. Molecular testing such as DNA sequencing or fragment analysis following PCR, and evaluation of copy number and gene positioning by FISH, have been developed and used clinically to identify mutations/fusions. Meanwhile, TKIs to target actionable mutations/fusions in several other oncogenes are being evaluated. High-throughput sequencing can provide therapy-predictive information as well as identify novel targetable genomic alterations. In this article, we report our experience enabling single-gene testing, and our evolution to panel-based next-generation sequencing.

Targeting the human epidermal growth factor receptor 2 pathway in breast cancer.

The discovery of amplification of human epidermal growth factor receptor 2 (HER2), a member of the epidermal growth factor receptor family, was an important milestone in our understanding of the biology of breast cancers. This heralded the discovery of trastuzumab, a humanized monoclonal antibody targeting HER2. Trastuzumab is the foundation of treatment of HER2-positive breast cancers, demonstrating dramatic responses in patients with metastatic disease. Unfortunately, most tumors will inevitably develop resistance to trastuzumab, necessitating the need for alternate HER2-directed therapeutic approaches. Recent advances in our understanding of the interaction between HER2 and other members of the epidermal growth factor receptor family have led to identification of newer agents, resulting in the expansion of the clinical armamentarium of available agents for the treatment of HER2-positive tumors. In this article, we review the molecular biology of the ERbb receptor family, the use of HER2-targeted agents in early and advanced breast cancer, and the next-generation anti-HER2 agents that are currently in clinical evaluation.

A comparison of imputation procedures and statistical tests for the analysis of two-dimensional electrophoresis data.

BACKGROUND: Numerous gel-based softwares exist to detect protein changes potentially associated with disease. The data, however, are abundant with technical and structural complexities, making statistical analysis a difficult task. A particularly important topic is how the various softwares handle missing data. To date, no one has extensively studied the impact that interpolating missing data has on subsequent analysis of protein spots. RESULTS: This work highlights the existing algorithms for handling missing data in two-dimensional gel analysis and performs a thorough comparison of the various algorithms and statistical tests on simulated and real datasets. For imputation methods, the best results in terms of root mean squared error are obtained using the least squares method of imputation along with the expectation maximization (EM) algorithm approach to estimate missing values with an array covariance structure. The bootstrapped versions of the statistical tests offer the most liberal option for determining protein spot significance while the generalized family wise error rate (gFWER) should be considered for controlling the multiple testing error. CONCLUSIONS: In summary, we advocate for a three-step statistical analysis of two-dimensional gel electrophoresis (2-DE) data with a data imputation step, choice of statistical test, and lastly an error control method in light of multiple testing. When determining the choice of statistical test, it is worth considering whether the protein spots will be subjected to mass spectrometry. If this is the case a more liberal test such as the percentile-based bootstrap t can be employed. For error control in electrophoresis experiments, we advocate that gFWER be controlled for multiple testing rather than the false discovery rate.

Cocaine and beta-blockers: the paradigm.

Cocaine is one of the most commonly used substances of abuse. The use of beta-blockers in cocaine induced acute coronary syndrome has long been a matter of debate. While it is widely believed that beta-blockers are contraindicated in cocaine toxicity, there appears to be some recognizable role for certain beta-blockers in ameliorating the cardiovascular as well as central nervous system effects of cocaine. This article explores the role of beta-blockers in the management of cocaine toxicity.

Minimizing variability in two-dimensional electrophoresis gel image analysis.

For reliable protein identification and quantitation, it is important to minimize the variability associated with two-dimensional electrophoresis (2-DE) analysis. Since experimental factors contribute largely to the variability observed in 2-DE, most studies have focused on reducing this variability with modest concern to the variability associated with post-experimental analyses. Although often ignored, software analyses of 2-DE gel images present a considerable source of variability in the analysis of proteins. In particular, cropping of gel images prior to quantitative 2-DE analysis has been shown to contribute a significant amount of variability in image analysis. To address this problem, we propose a simple, reliable, and objective method of cropping 2-DE gel images to consequently minimize the variability in 2-DE analysis.

Evaluating peptide mass fingerprinting-based protein identification.

Identification of proteins by mass spectrometry (MS) is an essential step in proteomic studies and is typically accomplished by either peptide mass fingerprinting (PMF) or amino acid sequencing of the peptide. Although sequence information from MS/MS analysis can be used to validate PMF-based protein identification, it may not be practical when analyzing a large number of proteins and when high- throughput MS/MS instrumentation is not readily available. At present, a vast majority of proteomic studies employ PMF. However, there are huge disparities in criteria used to identify proteins using PMF. Therefore, to reduce incorrect protein identification using PMF, and also to increase confidence in PMF-based protein identification without accompanying MS/MS analysis, definitive guiding principles are essential. To this end, we propose a value-based scoring system that provides guidance on evaluating when PMF-based protein identification can be deemed sufficient without accompanying amino acid sequence data from MS/MS analysis.

Effects of chronic ethanol administration on brain protein levels: a proteomic investigation using 2-D DIGE system.

The effects of chronic ethanol treatment on the brain proteome were investigated in the long-fin striped strain of zebrafish Danio rerio. Prolonged exposure to 0.5% (v/v) ethanol resulted in the development of tolerance to the ethanol-induced disruption of normal swimming behavior. This behavioral tolerance was manifested after two weeks of continuous treatment and was maintained for an additional three weeks. After four weeks of ethanol treatment, zebrafish brains were divided into 40,000 g supernatant and pellet fractions, and an Ettan 2-D fluorescence difference gel electrophoresis (DIGE) system was used to detect ethanol-induced alterations in the level of protein expression. Protein identification was carried out using matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry and the Mascot and ProFound search engines. In the present study, we have identified some novel protein targets as well as substantiated some putative previous targets of chronic ethanol exposure.

Distribution of ABO blood groups in acute leukaemias and lymphomas.

We studied the distribution of ABO blood groups in Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukaemia and acute lymphoblastic leukaemia, in children up to the age of 12 years, in a hospital-based retrospective study. Blood group data were recorded from the case records of all the patients in a tertiary care centre with the diagnosis of Hodgkin's lymphoma, non-Hodgkin's lymphoma, acute myeloid leukaemia and acute lymphoblastic leukaemia, during the period 1987-1997. There were 63 Hodgkin's lymphoma, 78 non-Hodgkin's lymphoma, 116 acute myeloid leukaemia and 522 acute lymphoblastic leukaemia patients. We assessed the distribution of ABO blood groups and the difference in the distribution from the source population. In Hodgkin's lymphoma, there were 45.6% [95% confidence interval (CI): 6.8-84.5] more patients with B blood group. In acute lymphoblastic leukaemia, there were 14.3% (95% CI: 3.2-25.2) more patients with O blood group. In Hodgkin's lymphoma and non-Hodgkin's lymphoma patients, there were 56.5% (95% CI: 19.9-85.4) and 52.9% (95% CI: 18.1-82.6) less patients with A blood group, respectively. This shows that the relationship between the ABO blood groups and haematological malignancies merits further investigation in a population-based prospective study. This is the first study of its kind in any Indian population.